| Phosphate is one of the main elements which plant growth needed, however, above 80% phosphate in soil is insoluble and unavailable for the plant. Phosphate solubilizing bacterium (PSB) can convert insoluble P into soluble form in the plant rhizosphere. Recently, PSB was gradually utilized in agriculture. In this paper, a high effective phosphate solubilizing bacterium was screened from the soil, its fermentation process was optimized and its application on maize planting was evaluated.There were 55 strains were screened from the soil of suburban area of Wuhan, which could grow in insoluble phoaphate medium, among them,36 strains had obvious transparent circle. A high effect phosphate solubilizing bacteria—L13 was obtained, with the ability of phytate solubilization 208.27 mg/L and calcium phosphate solubilization 449.03 mg/L. After that, strain L13 was identified as pseudomonas putida by biochemical characteristics and partial sequencing analysis of the 16S rDNA.The characteristics of phosphate solubilizing of p. putida L13 was investigated in PKO liquid medium, the result showed there was a good correlation beween the variable of phosphate solubilizing and cell growth. The phytase enzyme was mainly synthesized in log phase. Four kinds of known organic acids were analized in the broth of p. putida L1 3, which were oxialic acid, lactic acid,α-ketoglutarate acid and fumaric acid. The pH of broth decreased to 4.0 after two day’s growth, and then maintains it as far as the end. It is speculated the phytase and organic acid played an important role in log phase of cell growth, while the H+ releasing was the key factor in station phase for phosphate solubility. A slight increase of soluble P in decline phase of cell growth was owened to cell autolyzing.The medium and the culture conditions of p. putida L13 have been carried out by using BBD and one at a tiome method in flasks level, and furher validated in 3 L bioreactor. The optimized process was:glucose 17.64 g/L, yeast extract 2.39 g/L, (NH4)2SO4 1.41 g/L and buffer 101.53%, MgSO4.7H2O 0.3 g/L and KCl 0.1 g/L; initial pH 7.3, inoculum 0.5%, temperature 28℃and liquid volume in flask 30 mL/250 mL The results showed the biomass increased 16.70 times than the initial medium, and it reached 105.73×108CFU/mL. This study resolved the problem of wall-adherent growth and death in traditional fermentation medium of the phosphate solubilizing bacteria. The survival of p. putida L13 with peat, wheat bran, rapeseed meal and rice bran as the microbial agent carrier was studied. The result showed the peat was the best one for p. putida L13 preparation. After 90 d storage in room temperature, the biomass was still upon 1.0×108 CFU/g, no fungi contaminated and bacteria pollution rate was lower than 30 percent.It was found that five times pure strain dilution suspends can improved maize seed germination significantly, and 30 percent increased than control. It was also showed the broth dilution have a good improving effect for maize seed root length and root numbers. The uitilization of p. putida L13 combined with organic phosphte fertilizer and inorganic phosphate fertilizer was finished further. P. putida L13 was inoculated in maize rootsphere with composted pig manure and three kinds of inorganic phosphate fertilizers. The results showed that the content of soluble P and the activities of enzymes of soil with pig manure were higher during the maize growth phase. Compared with control, the plant hight, stem width, root length, fresh weight, dry weight and total P content of leaves of maize (growed 50 d) had obvious increased with p. putida L13 treatment. The experiment of three kinds of inorganic P fertilizers compouned with p. putida L13 showed that the order of maize growth promoting effect was:Calcium dihydrogen phosphate> rock phosphate> tricalcium phoaphate, Owing to the lower cost of the phosphate rock, it was advised for further application togher with p. putida L13 in agriculture. |
PDF Full Text Request